We have investigated the oligomeric properties of procaspase-3 and a mutant that lacks the pro-domain (called pro-less variant). In addition, we have examined the interactions of the 28 amino acid pro-peptide when added in trans to the pro-less variant. By sedimentation equilibrium studies, we have found that procapase-3 is a stable dimer in solution at 25 degrees C and pH 7.2, and we estimate an upper limit for the equilibrium dissociation constant of approximately 50 nM. Considering the expression levels of caspase-3 in Jurkat cells, we predict that procaspase-3 exists as a dimer in vivo. The pro-less variant is also a dimer, with little apparent change in the equilibrium dissociation constant. Thus, in contrast with the long pro-domain caspases, the pro-peptide of caspase-3 does not appear to be involved in dimerization. Results from circular dichroism, fluorescence anisotropy, and FTIR studies demonstrate that the pro-domain interacts weakly with the pro-less variant. The data suggest that the pro-peptide adopts a beta-structure when in contact with the protein, but it is a random coil when free in solution. In addition, when added in trans, the pro-peptide does not inhibit the activity of the mature caspase-3 heterotetramer. On the other hand, the active caspase-3 does not efficiently hydrolyze the pro-domain at the NSVD(9) sequence as occurs when the pro-peptide is in cis to the protease domain. Based on these results, we propose a model for maturation of the procaspase-3 dimer.
Dihydrofolate reductase (DHFR) from Escherichia coli does not interact with the molecular chaperonin GroEL regardless of whether the interaction is initiated from the native or the unfolded state. In contrast, murine DHFR shows a strong interaction with GroEL. Using the structure of human DHFR as a model for the murine protein, a superimposition of the two structures shows that there are three distinct external loops in the eukaryotic DHFR that are not present in the E. coli protein. Removal of one loop (residues 99-108) from the eukaryotic murine DHFR has no effect on the interaction with GroEL. On the basis of the differences in structures, we inserted either of two surface loops of murine DHFR into the corresponding regions of E. coli DHFR. In the first mutant (EcDHFR-i(9)36), residues 36 and 37 (L-N) of E. coli DHFR were replaced with the nine amino acid sequence T-T-S-S-V-E-G-K-Q. In the second mutant (EcDHFR-i(7)136), residues 136-139 (V-F-S-E) of E. coli DHFR were replaced with the seven amino acid sequence L-P-E-Y-P-G-V. Both E. coli DHFR mutants formed a complex with GroEL starting from either the native or the unfolded states of DHFR. The binding was specific since the presence of MgATP caused the release of the proteins from GroEL. As with murine DHFR, nonnative conformations of EcDHFR-i(9)36 and EcDHFR-i(7)136 are bound to GroEL. Fluorescence titration techniques were used to quantitate the interaction between GroEL and these proteins. A simple chromatographic procedure was developed to remove contaminating tryptophan containing peptides from GroEL samples. The mutant EcDHFR-i(7)136 binds to GroEL with a stoichiometry of 4-5 mol of DHFR per mol of GroEL tetradecamer, while murine DHFR binds to GroEL with a stoichiometry of 2 mol of DHFR per mol of GroEL tetradecamer. Both murine DHFR and EcDHFR-i(7)136 bind to GroEL very tightly, with equilibrium dissociation constants of less than 85 nM.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.